Molecular Structure,physicochemical Properties And Synthetic Mechanism Of Dextrans Produced By Lactic Acid Bacteria

Lactic acid bacteria(LAB)are recognized as beneficial microorganisms used in the production of a variety of fermented foods.The extracellular polysaccharides(EPS)produced by the bacteria can be added to food formulations as additives,thickeners and emulsifiers and also have anti-tumor and anti-ulcer activity in addition to promoting health by acting as prebiotics,lowering cholesterol,and activating the immune system.Although LAB EPS have shown promise for various applications,current understanding and research remain limited.In this study,two LAB strains producing high yields of EPSs were isolated from fermented foods,and the molecular structures,physicochemical properties,biological activities and synthetic mechanisms of their EPSs were studied.The two LAB strains,dubbed L3 and PC,were isolated from sausage and homemade sauerkraut,respectively.They showed obvious sugar production when cultured on MRS-S plates,indicative of EPS production.Based on morphology,physiological and biochemical indexes,and 16S r DNA sequencing,L3 was identified as Lactobacillus sakei,with a 16S r DNA sequence 99%homologous to Lac.sakei KLDS 1.0729.PC was identified as Leuconostoc pseudomesenteroides,with a 16S r DNA sequence 99%homologous to Leu.pseudomesenteroides BFE 6644.Subsequently,the medium and culture conditions affecting EPSs yields of L3 and PC were optimized.After optimization by single factor experimentation,the yield of L3 EPS increased from 38.05±1.14 g/L to 64.93±0.80 g/L.Plackett-Burman(PB)experiments manifested that the significant factors affecting the yield of L3 EPS were sucrose concentration,initial p H and inoculum and that sucrose concentration had the greatest impact on final yield.Based on these results,we performed a central composite design(CCD)experiment.The maximum L3 EPS yield predicted by the model was68.28 g/L,achieved when the sucrose content was 127.80 g/L,the initial p H was 6.87,and the inoculum was 3.15%(v/v).Experimental results were consistent with the predicted values.In addition,we increased the EPS yield for strain PC from 35.13±0.61g/L to 62.07±1.42 g/L through single-factor,PB,steepest ascent and CCD optimization experiments.The two strains were cultured using the optimized medium and culture conditions,and EPSs were separated and purified from the cultures in order to study their molecular structures.Purification was carried out via centrifugation,95%(v/v)alcohol precipitation,protein removal by trichloroacetic acid,dialysis and Sephadex G-100 gel filtration chromatography.The results of gas chromatography,fourier transform infrared spectroscopy and nuclear magnetic resonance showed that the EPSs isolated from the two strains were both dextrans composed of repeating?6)?-D-Glcp-(1?units.The molecular weights of the L3 and PC EPSs were determined by high performance size exclusion chromatography(HPSEC)to be 3.25×10~6 and 3.13×10~6 Da,respectively.Scanning electron microscopy analysis showed that the L3 EPS polymer had a porous and branched morphology,while the PC purified EPS polymer consisted of irregular flakes with a porous structure.Atomic force microscopy results showed round or spike-like lumps on both EPSs polymer surfaces.Congo red experiments,circular dichroism spectroscopy and?-elimination reactions indicated that both EPSs comprise a single strand of irregular coils without a regular triple helix structure and showed an O-form linkage type for the glycopeptide bonds.Next,the physical-chemical properties and biological activities of the two EPSs were analyzed.The degradation temperatures of L3 and PC EPSs were 272 and 304.9°C,respectively,and the melting endothermic peak temperatures were 210.8 and 236.5°C,respectively.Meanwhile,L3 and PC EPSs have high hydrophilicity.The average particle size and Zeta potential were measured for solutions of the two EPSs at different concentrations.Results showed that when concentration increased from 0.125 to 2mg/m L,the Zeta potential of PC EPS solutions decreased from-2.7±0.1 m V to-7.3±0.2m V,while the average particle size increased from 220.1±1.9 nm to 332.8±5.3 nm.For solutions of L3 EPS,the Zeta potential decreased from from-1.9±0.2 m V to-8.3±0.1m V,while the average particle size increased from 217.5±0.7 nm to 251.1±0.4 nm.At room temperature,the intrinsic viscosities of the L3 and PC EPSs in water were 202.59and 238.27 m L/g,respectively.The results of experiments to determine rheological properties showed that the apparent viscosity of the two EPSs increased with increasing concentration and decreasing temperature and p H within a certain range.In terms of emulsifying activity,aqueous solutions of 2 mg/m L L3 and PC EPSs showed the highest emulsifying activity for sunflower seed oil(62.30±0.06%)and soybean oil(57.57±0.13%),respectively.Milk coagulation experiments showed that L3 and PC were able to completely solidify skim milk containing 12%(w/v)and 9%(w/v)sucrose,respectively.To test probiotic properties,the effect of the EPSs addition on the in vitro growth of Lac.delbrueckii,Streptococcus thermophilus,Lac.casei,Lac.plantarum and Lac.paracasei were examined.For L3 EPS,the growth-promoting effect was greatest for S.thermophilus;addition of 2 g/L of L3 EPS shortened the lag phase by 4 hours and increased the maximum biomass attainable by 5.77%.For PC EPS,the growth-promoting effect was greatest for Lac.plantarum;addition of 2 g/L PC EPS shortened the lag phase by 9 hours and increased the maximum biomass attainable by 11.89%.In order to explore the molecular mechanisms of EPS production,we selected the L3 strain for study and sequenced its transcriptome in MRS medium with(MRS-S)and without(MRS)sucrose supplementation,corresponding to conditions fostering high and low levels of EPS production,respectively.The results showed significant differences in gene expression between the two conditions,with 230 genes significantly up-regulated and 196 significantly down-regulated in MRS-S medium compared to MRS medium.Among these differentially expressed genes,genes related to UMP,fatty acid and folic acid metabolism were down-regulated to varying degrees,while enzymes related to sucrose transport and metabolism were generally up-regulated.q RT-PCR results confirmed the trends seen in the transcriptomics data.Transcript levels of sucrose transporter,sucrose-6-phosphate hydrolase and fructose transporter were increased by 28.62±2.56,31.20±0.78 and 74.76±14.25 fold in the L3 strain grown on MRS-S medium.Glucansucrase,key for the synthesis of glucan from sucrose in LAB,was up-regulated by 24.19±4.29-fold in the L3 strain grown on MRS-S medium.The above findings provided a clear basis for elucidating the mechanism of EPS synthesis by strain L3.Based on these results,we modeled interactions of glucansucrase,sucrose transporter,sucrose-6-phosphate hydrolase and fructose transporter with their respective substrates through homology modeling and molecular docking technology.The results showed that these four proteins formed pockets at specific amino acid residues and could interact with sucrose or fructose via Van der Waals forces or hydrophobic interactions,thus enabling enzymatic catalysis or transmembrane transport.In summary,this work reported the isolation of two novel LAB strains that produced EPSs and various characteristics of the EPSs purified from these strains.Both strains produced dextran,and the study partially elucidated the biosynthetic mechanism of dextran by one of the strains.The two EPSs exhibited physical-chemical properties and biological activities that suggested potential value for various applications.All together,our study provided a basis for further research on and application of LAB EPS.